Step by patient step, one man is drawing ever closer to the real Da Vinci mystery: tracking down the master’s greatest painting, lost for four and a half centuries. And it is hidden, he believes, in a room at the heart of political power since the Middle Ages in Florence.

For art historians, finding Leonardo’s lost Battle of Anghiari is in the same league as finding the Titanic or the still lost tomb of the Ancient Egyptian architect Imhotep — as big as you can get. The man who believes he is on the verge of this discovery is Maurizio Seracini, now striding towards me in the Piazza della Signoria, home of Italy’s “Louvre” — the Uffizi gallery. But Dr Seracini’s real passion lies not in the square but deep inside the looming shape of the Palazzo Vecchio flanking it. As The Da Vinci Detective, a Channel Four documentary to be aired this week, reveals, he believes that hidden behind a mural in the Sala del Gran Consiglio lies one of the most famous works of art in the world.

The Battle of Anghiari is known through numerous copies produced by admiring artists, and its fame rests on the fact that it was not only one of the greatest tests of Leonardo’s skills and his largest and most substantial work, but also that it was a contender in the most intense art competition of the Renaissance.

Dr Seracini has spent more than 30 years on his quest, and his detective methods have included a combination of the most advanced technologies. But it has earned him calumny and contempt from the scholarly art world and heritage lobby, some of them pursuing rival investigations.

The painting was commissioned in 1504 by Piero Soderini, head of the Republican government of Florence, to commemorate the Republic’s military victory in 1440 over the hated Milanese on the plains of Anghiari. It was intended to stand as a powerful statement of Florentine independence and was to stand opposite another painting, showing the Battle of Cascina, an older Republican triumph, this time over Pisa in 1364. The artist of this work was to be none other than Michelangelo.

The two men had an intense dislike for one another. The person signing Leonardo’s contract was Niccolo Machiavelli, Secretary of the Republic, whose name now stands for political cunning.

When both artists hung up their cartoons — large scale drawings showing the intended scenes — Michelangelo's composition focused on a group of nude soldiers bathing while Leonardo’s centred on a furious battle between horsemen in which the sheer beauty of the horses takes centre stage.

In so doing, he was faithfully following the instruction given to him by his patron to represent a key moment in the Battle of Anghiari, the fierce Fight for the Standard, that witnesses recall as its turning point. Giorgio Vasari’s breathless description of Leonardo’s painting gives a sense of its power: “It would be impossible to express the inventiveness of Leonardo’s design for the soldiers’ uniforms, which he sketched in all their variety, or the crests of the helmets and other ornaments, not to mention the incredible skill he demonstrated in the shape and features of the horses, which Leonardo, better than any other master, created with their boldness, muscles and graceful beauty.”

The great scheme, however, was overtaken by events. In March 1505 Michelangelo left for Rome to work on the tomb of the great art patron Pope Julius II, never to finish the Battle of Cascina. Perhaps sensing that this was now his moment, Leonardo began painting his Battle later that year. Right from the start, however, disaster dogged his efforts.

Dr Seracini opens his briefcase and pulls out a copy of Leonardo’s diary entry for June 6: “Just as I lowered the brush, the weather changed for the worse and the bell started to toll . . . the cartoon was torn, water poured down and . . . it rained very heavily until nightfall and the day was as night.”

Further catastrophe came when Leonardo was using lighted braziers to warm the room to speed the painting’s drying process. As the braziers got hotter, the painting started to drip down the wall. “Vasari says this was because of Leonardo’s technical mistakes,” Dr Seracini explains, “and that he used wax in the paint presumably to create certain effects, but it could also have been that wax was used to seal the plaster underneath the paintings and that this liquidised. But it’s also the case that he was given very cheap materials to work with. We have a list of all his ingredients. I’d like to do a mock up and test what happens.”

When the Republic fell with the return of the Medici, it was Vasari who was commissioned in 1563 to redesign the chamber and to create six murals celebrating Medici military victories to obliterate those commissioned by the Republic. In the process, Dr Seracini believes, Vasari covered up the Battle rather than destroy it out of recognition of its outstanding qualities. “He’d done this before when he altered Sta Maria Novella [a Florentine church] and protected the Holy Trinity by Masaccio from destruction.” As if confirming his hunch, he’s discovered a small inscription written on a flag at the top of one of Vasari’s murals with the words cerca trova — “seek and ye shall find”.

Dr Seracini takes me to the window through which he believes the rain would have come in, pointing out that when you look at the copies of the Battle, the light source naturally comes from the right, just where the window is. He gestures down the long wall that runs back into the huge hall. “There’s a contemporary account that says that Leonardo’s paintings stood above where the 12 men of the senior council sat and I think we now know where that is,” he says.

Supported by recent archival research conducted by Professor Rab Hatfield, of Syracuse University’s department in Florence, into the architectural changes the hall has undergone over the past 500 years, Dr Seracini believes he has found the exact spot where the Battle once stood.

His journey to this point, during which his dedication to his quest has cost him his marriage, has been anything but easy. Originally an engineering student at San Diego University, he studied art history as a hobby and in 1975 was taken on by Professor Carlo Pedretti, a Leonardo expert keen to find the location of the Battle. Back then he lacked the advanced technical apparatus at his disposal today, but now he has a battery of sophisticated devices.

In his studio the other side of the River Arno, Dr Seracini shows me image after image of the Vasari mural under which he thinks the Battle still lies and Leonardo’s iconic Adoration of the Magi, held by the Uffizi gallery. His approach — unique in the art world — is to combine X-ray, X-ray fluoroscopy, adapted ultrasound, thermography and other non-invasive investigative processes to explore the structure of walls, paintings, ceilings and gaps — to ascertain whether the Battle really does lie beneath. X-rays, for example, reveal the presence of pencil lead.

Dr Seracini has already caused considerable upset at the Uffizi by demonstrating through analysis of paint layers that most of the Adoration isn’t by Leonardo. His remarkable photographs have revealed similarities between Leonardo’s hitherto invisible under-drawing, in which a fierce battle between horsemen is evident, and the composition of the same scene from the Battle. Using the same processes to examine the Vasari mural, Dr Seracini has found a 1? in gap behind it. Could the Battle lie underneath and be undamaged? “Why not?” he asks. “Another test I would do would be to bombard the wall with low-level nuclear radiation, and by counting the gamma rays which would bounce back, we would then be able to detect the presence of pigment behind the plaster.” That would surely worry the authorities. (The Palazzo Vecchio, keen to beat him, conducted its own tests on what turned out to be the wrong part of the mural.) The ultimate proof would be to remove the Vasari mural and conduct tests on the supposed Battle canvas beneath, though this would be controversial — you are endangering one work of art in the cause of another.

Dr Seracini isn’t keen on making such a dramatic step — he wants to amass as much proof as possible by non-invasive techniques. But do not doubt his seriousness and ambition. “It’s becoming a little clearer to people what the role of science could be and what it should become.